Lack of Serological and Molecular Association between Toxoplasma Gondii Exposure and Obesity: A Case-Control Study.

The association between T. gondii infection and obesity has been scantly studied. Through an age-, and gender-matched case-control study, we determined the association of T. gondii infection and obesity using serological and molecular methods. Cases included 203 persons with obesity, and controls included 203 persons without obesity. Participants were tested for the presence of anti-Toxoplasma IgG antibodies using an enzyme-linked immunoassay (EIA). IgG seropositive individuals were further tested for the presence of anti-T. gondii IgM antibodies using an EIA, and T. gondii DNA by polymerase chain reaction (PCR). Anti-T. gondii IgG antibodies were found in 16 (7.9%) of the 203 cases and in 18 (8.9%) of the 203 controls (OR=0.87; 95% CI: 0.43-1.77; P=0.72). One (6.3%) of the 16 anti-T. gondii IgG seropositive cases and 6 (33.3%) of the 18 anti-T. gondii IgG seropositive controls were positive for IgM (P=0.09). Mean body mass index (35.5 ± 4.5) in T. gondii seropositive cases was similar (P=0.57) to that (36.1 ± 4.5) found in T. gondii seronegative cases. Stratification by obesity classes (I, II, and III) did not reveal differences (P>0.05) in seroprevalences (7.8%, 7.9%, and 8.1%, respectively) or high (>150 IU/ml) IgG antibody levels (3.3%, 3.9%, and 2.7%, respectively). PCR was positive in 5 (31.3%) of 16 cases, and in 5 (27.8%) of 18 controls examined (P=1.0). We found no serological or molecular evidence of an association between T. gondii infection and obesity in people attending a public health center in the northern Mexican city of Durango.


INTRODUCTION
The coccidian parasite Toxoplasma gondii (T. gondii) is a common pathogen with worldwide distribution (1). This parasite is usually transmitted to humans by ingestion of food or water contaminated with oocysts shed by cats, and ingestion of raw or undercooked meat containing tissue cysts (2,3). In addition, primary infection with T. gondii in pregnant women may lead to vertical transmission with risk for congenital disease (4). Infection with T. gondii is usually asymptomatic, however, this infection may lead to disease of the central nervous system, eyes and lymph nodes (5,6). In immunocompromised individuals, a reactivation of T. gondii infection may result in a severe and life-threatening disease with involvement of the central nervous system (2,7). Infection with T. gondii has been associated with changes in behavior in humans and animals (8,9). An increase in dopamine production induced by T. gondii may contribute to behavioral changes (8). Several psychiatric disorders have been linked to infection with T. gondii including schizophrenia (10), bipolar disorder, and obsessive-compulsive disorder (11).
Obesity is a major health problem, and its prevalence is high in many parts of the world (12). In Mexico, more than 50% of adults have overweigh and obesity (13). Obesity and overweight have been linked to important causes of mortality in Mexico including coronary heart disease, type-2 diabetes mellitus, cancer, and stroke (13). Infection with T. gondii might be associated with obesity because this infection is usually acquired by food. Obese people may eat in a higher quantity than non-obese people; and therefore, this increase in eating might increase the risk of consuming food likely contaminated with T. gondii. It is possible that consumption of a double portion of meat (a well-known source of T. gondii) might increase twofold the risk for acquiring infection. It is also possible that drinking untreated water or consuming unwashed raw vegetables or fruits in high quantities might also increase the risk for T. gondii infection. The association of T. gondii infection and obesity has been scantly studied. Reeves and coworkers (14) found an association between positive serology to T. gondii and obesity in psychiatrically healthy adults. Rubicz and coworkers (15) found a 9% seroprevalence of T. gondii infection in Mexican Americans from San Antonio, Texas that suffered from high rates of obesity and type-2 diabetes. In contrast, in a multinational epidemiological study of individuals from Iceland, Sweden and Estonia, no association of anti-T. gondii IgG antibodies and body mass index was found (16). However, in a recent study in Germany a body mass index ≥30 was an independent risk factor for IgG seropositivity to T. gondii (17). The present study therefore aimed to determine whether T. gondii infection is associated with obesity in adults attending a public clinic of family medicine in Durango City, Mexico. Determining this association may help for an optimal planning of preventive measures against T. gondii infection.

Selection and description of participants
Through an age-and gender-matched case control study design, we studied 203 individuals with obesity and 203 individuals without obesity attended in a public clinic of familiar medicine in Durango City, Mexico. This study was performed from June 2015 to August 2016. Inclusion criteria for enrollment of cases were: 1) individuals with obesity attending a public primary health care center (Clinic of Family Medicine, Institute of Security and Social Services of State Workers) in Durango City, Mexico; 2) aged 18 years and older; and 3) who accepted to participate in the study. Socioeconomic status and occupation were not restrictive criteria for enrollment.
Obesity was defined as a body mass index ≥30; and classified in class I, class II, and class III when body mass indexes were 30-34.9, 35.0-39.9, and ≥40.0, respectively (13). Control subjects were matched with cases for age and gender. Cases included 42 (20.7%) males and 161 (79.3%) females, and their mean age was 51.4 ± 11.6 (range 22-83) years old. Controls were randomly selected. Inclusion criteria for enrollment of controls subjects were: 1) individuals without obesity attending the same public primary health care center where cases were selected; 2) aged 18 years and older; and 3) who accepted to participate in the study. Controls included 42 (20.7%) males and 161 (79.3%) females. Mean age in control subjects were 51.5 ± 11.5 (range 20-80) years old. No statistically significant difference (P=0.89) in age between cases and controls was found.

Technical information
Sera from cases and controls were obtained and kept frozen at -20°C until analyzed. Anti-T. gondii IgG antibodies were detected in sera using the commercially available enzyme immunoassay (EIA) kit "Toxoplasma IgG" (Diagnostic Automation/Cortez Diagnostics Inc., Woodland Hills, CA, USA). Anti-T. gondii IgG antibody levels were expressed as International Units (IU)/ml. We used a cut-off of 8 IU/ml for seropositivity. All serum samples positive for anti-T. gondii IgG antibodies were further analyzed for anti-T. gondii IgM antibodies using the commercially available EIA "Toxoplasma IgM" kit (Diagnostic Automation/Cortez Diagnostics Inc.). Both IgG and IgM EIAs were performed following the manufacturer's instructions.
Cases and controls seropositive for Toxoplasma-specific IgG antibodies by EIA were further analyzed to detect DNA of T. gondii by nested-polymerase chain reaction (PCR). DNA was extracted from whole blood samples of cases and controls according to a protocol described by Iranpour and Esmailizadeh [http://www.protocol-online. org/prot/Protocols/Rapid-Extraction-of-High-Quality-DNA-from-Whole-Blood-Stored-at-4-C-for-Long-Period-4175.html]. PCR amplification was performed following the PCR protocol described by Roth et al (18). Primers directed against the B1 gene of T. gondii were used. PCR amplified material was analyzed by agarose gel electrophoresis, stained with ethidium bromide, and visualized by ultraviolet illumination.

Statistics
Data was analyzed using the software Epi Info 7 and SPSS 15.0 (SPSS Inc. Chicago, Illinois). We calculated the sample size using the following values: a 95% confidence level, a power of 80%, a 1:1 proportion of cases and controls, and a reference seroprevalence of 6.1% (19) as the expected frequency of exposure in controls, and an odds ratio of 2.8. Thus, a sample size of 195 cases and 195 controls was obtained. Age among cases and controls was compared with the student's t test. The association of T. gondii infection and obesity was analyzed with the twotailed Pearson's chi-squared test. We calculated the odds ratio (OR) and 95% confidence interval (CI), and statistical significance was set at a P value < 0.05.

Ethics aspects
The ethics committee of the Institute of Security and Social Services of State Workers in Durango City, Mexico approved this study. Participation in the study was voluntary, and a written informed consent was obtained from each participant.
With respect to detection of T. gondii DNA in whole blood of anti-T. gondii IgG positive participants, PCR was positive in 5 (31.3%) of 16 cases and in 5 (27.8%) of 18 controls examined. No statistically significant difference in the frequencies of T. gondii DNA positivity among cases and controls was found (P=1.0). Stratification by age and gender groups did not show differences (P>0.05) in seroprevalences among cases and controls (Table 1). T. gondii DNA was found in three cases with >150 IU/ml of IgG antibodies and in two cases with <100 IU/ml of IgG antibodies. All 5 cases with T. gondii DNA were negative to anti-T. gondii IgM antibodies. T. gondii DNA was found in three cases with obesity class I, in one case with obesity class II, and in one case with obesity class III.

DISCUSSION
Very little is known about the association of T. gondii infection and obesity. Results of a few studies about this association have shown conflicting results (14)(15)(16)(17). Positive association between seroprevalence of T. gondii infection and obesity has been found in adults in Germany (14,17). In contrast, a low (9%) seroprevalence of T. gondii infection in Mexican Americans from San Antonio, Texas that suffered from high rates of obesity and type-2 diabetes was found (15). In addition, no association of seroprevalence of T. gondii infection and body mass index was found in a multinational epidemiological study in Iceland, Sweden and Estonia, (16). Therefore, we sought to determine whether T. gondii infection is associated with obesity in adults attending a public clinic of family medicine in the northern Mexican city of Durango. For this purpose, we assessed not only the prevalence of anti-T. gondii IgG antibodies but also the IgG levels, anti-T. gondii IgM seropositivity, and detection of T. gondii DNA. Results of the present study indicate that anti-T gondii IgG and IgM seropositivity rates, IgG levels, and frequency of T. gondii DNA in obese people are similar to those observed in age-and gender-matched control subjects without obesity attended in the same clinic of family medicine. Therefore, our results based on serological and molecular methods do not support an association between obesity and T. gondii infection. Results of the present study agree with the lack of association between body mass index and T. gondii IgG seroprevalence found in a multinational epidemiological study of individuals from Iceland, Sweden and Estonia (16), and with the low (9%) seroprevalence of T. gondii infection found in Mexican Americans that suffered from high rates of obesity and type-2 diabetes reported by Rubicz and coworkers (15). In contrast, our results conflict with those reported in two German studies (14,17). A positive serology to T. gondii associated with obesity in psychiatrically healthy adults in Germany was reported by Reeves and coworkers (14). Furthermore, a body mass index ≥30 was an independent risk factor for IgG seropositivity to T. gondii in a nationwide representative cross-sectional study in Germany (17). It is not clear why the association of T. gondii infection and obesity was found in populations in Germany but not in obese people in the present study. It is likely that differences in the char-acteristics of the populations and study designs among the studies might explain the differences in the association. In the study of Reeves and coworkers (14), the association between T. gondii infection and obesity was observed in subjects 60 years and older but not in subjects younger than 60 years. Stratification by age groups in our study (<30, 31-50, and >50 years) did not show an association of infection and obesity. In addition, we used an age-and gender-matched case-control study design whereas Reeves and coworkers performed adjustment by age but not by gender. The number of obese participants in the study by Reeves and coworkers was 74 (14), whereas we studied 203 obese participants. On the other hand, in the study of Wilking and coworkers (17) who reported an association of T. gondii seropositivity and obesity, researchers studied a large number (1,023) of obese participants, but the study design was cross-sectional, and no adjustment or stratification by age for T. gondii seropositivity was performed. In addition, Wilking and coworkers (17) used an automatic enzyme-linked fluorescence assay for detection of anti-T. gondii IgG antibodies whereas we used a manual enzymelinked immunosorbent assay. We are not aware of a previous study about the association of T. gondii infection and obesity using molecular methods. However, in the present study using T. gondii PCR, no association between obesity and T. gondii DNA was found. The lack of association between obesity and T. gondii was unexpected since obese people may eat more than non-obese people; and therefore, an increase in eating might increase the risk of consuming food likely contaminated with T. gondii.
The limitations of the present study include the investigation of a relatively small cohort of obese people attending a single public health center. The socioeconomic sta-

CONCLUSIONS
We conclude that there is not serological or molecular evidence of an association between T. gondii infection and obesity in people attended in a public family medicine health center in the northern Mexican city of Durango. Further research to elucidate the role of T. gondii in obesity is needed.